The mechanism limiting the red cell life span will be investigated in detail on groups of red cells of progressively increasing age isolated by ultracentrifugation on discontinuous density gradients of arabinogalactan. The rates of metabolic changes with "in vivo" red cell aging will be computed by measuring progressive changes in the various cell functions. The overall effects on cell metabolism will be evaluated by studies of accumulation of intermediate products as well as by analysis of metabolic activities in cell lysates supplemented with substrates, cofactors, inhibitors and activators. Changes in kinetic characteristics of key enzymes as well as shifts in isozyme patterns will be evaluated. These data will clarify the complex interplay of several metabolic alterations and offer insight into the mechanism of molecular aging of enzymes. These studies will be extended to genetic disorders of red cell metabolism. The molecular subunit structure of normal red cell phosphofructokinase and pyruvate kinase will be clarified by purification, dissociation and recombination and chromatographic analysis of isozymes. The kinetic characteristics of individual isozymes will be defined. The effect of cell aging on kinetic characteristics will be studied. These techniques will be applied to the study of defective mutant enzymes and the findings correlated with the clinical syndromes. Normal and defective mutants of glucose-6-phosphate dehydrogenase will be studied with regard to their allosteric affinity for substrates and "in vivo" changes in sensitivity to redox effects. The technique of isopycnic centrifugation will be applied to blood cell separation for laboratory and eventual blood banking use. The relationship between platelet density, size and function will be evaluated, utilizing a rapid and precise microtechnique for platelet counting and sizing. The clinical significance of changes in platelet size distribution will be evaluated in various pathological conditions, including neonatal thrombocytopenia.